Project Abstract Vascular oxidative stress is strongly implicated in the pathogenesis of virtually all primary risk factors for cardiovascular diseases (CVD), a leading cause of death globally. Classic antioxidants (?-carotene, Vitamin A and E) aimed at scavenging the short-lived free radicals have limited benefits in patients with CVD. Development of different therapeutic approaches combating vascular oxidative stress appears critical. Protein S- glutathionylation (Pr-SSG), the prevalent form of oxidant-induced reversible post-translational modification, recently emerged as an important redox regulatory mechanism in CVD. We found that in endothelial cells (ECs) isolated from patients with type 2 diabetes mellitus, the level of Pr-SSG was significantly elevated. This important finding is further confirmed in a hyperlipidemic mouse model showing markedly increased Pr-SSG concomitant with a down-regulation of Glrx-1, a specific de-glutathionylation enzyme, in aortic atherosclerotic lesions, particularly in ECs. Transgenic overexpression of Glrx-1 in ApoE-/- mouse strain protects against diet-induced aortic endothelial hyper-permeability and attenuates atherosclerosis (AS) development. These exciting preliminary results lead to a central hypothesis of this grant proposal that EC Glrx-1 has a protective role in metabolic stress-induced EC dysfunction and AS. Specific Aim#1 will test in vivo the hypothesis that EC-specific up-regulation of Glrx-1 will attenuate AS progression by improving EC dysfunction caused by metabolic abnormalities using EC specific Glrx-1 transgenic mice with ApoE-/- background. Our redox proteomic and biochemical studies in metabolically stressed ECs identify Rac1 as a specific target of Glrx-1 and demonstrate inhibitory effect of Glrx-1 on proteolytic activation of sterol regulatory element binding proteins (SREBPs), the central players in lipid homeostasis and EC dysfunction. Therefore, specific Aim#2 will test in vitro the novel hypothesis that Glrx-1 improves EC function through redox regulation of Rac1/SREBP signaling, employing comprehensive approaches including primary ECs from Glrx-1 TG and KO mice, pharmacological and genetic means (siRNA and adenovirus encoding Rac1 wild type and redox-resistant mutants), and redox biochemical techniques (Biotin-switch assay and immunodetection of Pr-SSG). Specific Aim#3 will test in vivo the hypothesis that supplementation of vascular ECs with Glrx-1 gene and/or recombinant protein can reverse vascular dysfunction and retard AS employing endothelium-targeted liposomal drug delivery system. We believe that the proposed studies will provide novel information about how Glrx-1 and glutathionylation of Rac1 are involved in EC dysfunction and AS complicated in metabolic disorders, and will seek to establish Glrx-1 as a prospective therapeutic agent for vascular injury in the setting of diabetes.